polymer hybrid films: nanoconstruction via specific recognition

PII: S0968-5677(98)00024-8

Supramolecular Science 5 (1998) 309—315  1998 Published by Elsevier Science Limited Printed in Great Britain. All rights reserved 0968-5677/98/$19.00

Layer-by-layer assembled protein/polymer hybrid films: nanoconstruction via specific recognition T. CassierR and K. Lowack n Institut fu¨ r Physikalische Chemie, Universita¨ t Mainz, Welder Weg 11, D-55099 Mainz, Germany

G. Decher m Universite´ Louis Pasteur and C.N.R.S., Institut Charles Sadron, 6, rue Boussingault, 67083 Strasbourg Cedex, France In the present study it is shown that streptavidin-containing multilayer films with varying numbers of polyelectrolyte spacer layers can be fabricated reproducibly using optimized deposition conditions. Direct alternation of streptavidin and PLB leads to multilayer systems with an average streptavidin thickness of 5.3 nm, which is in good agreement with the dimensions of the protein. When the streptavidin layers are spacered by more polyelectrolyte layers, the distance between the protein sheets is increased up to, e.g. 6.5 nm in the case of (PLB/PSS/PAH/PSS/PLB) as spacer layer. X-ray reflectivity reveals that streptavidin increases the surface roughness of the films, probably due to the rigid three-dimensional structure of the protein. The control of surface roughness seems to be essential for a successful multilayer build-up. The property of PLB to provide for multilayer construction by two different interactions (electrostatic and specific) allowed to probe the interpenetration depth of adjacent layers. For the [PLB/(PSS/PL)/streptavidin] system an interpenetration depth of about 4 polymer layers corresponding to approximately 3.4 nm has been derived. These data are in agreement with a model for pure polyelectrolyte films obtained from neutron and X-ray reflectivity data.  1998 Published by Elsevier Science Limited. All rights reserved. (Keywords: Specific recognition; multilayer; polyelectrolyte adsorption; self-assembly; streptavidin; biotin)

INTRODUCTION In the early 1990s we have reported on a method to fabricate multilayer assemblies by consecutive adsorption of positively and negatively charged polyelectrolytes. While it has been relatively easy to include proteins into such films via electrostatic interaction\, the use of specific interactions for protein incorporation is somewhat more challenging owing to the high steric demands of this interaction. The specific adsorption of proteins to a given surface requires the modification of this interface with functional entities in such a way that they are accessible to the protein. We have chosen the well-established biotin/streptavidin system for these experiments, because the carboxylic group of biotin can easily be reacted with, e.g. aminogroups by maintaining its high binding constant to streptavidin of 10 l mol\. With respect to topology, streptavidin is a very interesting protein because it offers four binding sites, two of which are located at opposite ends of the molecule. In the case of multilayer films that are formed both by electrostatic attraction and by specific recognition, a bifuncRPresent address: Max-Plank-Institut fu¨r Polymerforschung (MPI-P), Ackermannweg 10, Postfach 3148, D-55021 Mainz, Germany m To whom correspondence should be addressed n Present address: Institut fu¨r Mikrotechnik Mainz (IMM), Carl-ZeissStr. 18—20, D-55129 Mainz-Hechtsheim, Germany

tional polymer is required as mediator molecule that can bind to a charged surface via its ionic groups of opposite charge and that exposes the groups to which the protein can bind towards the solution site of the newly adsorbed layer. We have selected poly(L-lysine) (PL) as polycation because it is readily available and because the reaction of reactive esters of biotin with the e-aminogroups of the lysine residues in proteins is already widely used. So by a simple polymer analog reaction one obtains a statistical copolymer poly(L-lysine-co-e-biotinyl-L-lysine) (PLB) which fulfills all necessary prerequisites for binding as mentioned above. From earlier experiments with fluorescently labeled streptavidin, we already know that streptavidin is specifically bound to PLB and that unspecific binding of streptavidin to the non-biotinylated poly(Llysine) is, at large, negligible. In 1993 we have preliminary reported on the alternation of poly(L-lysine-co-e-biotinyl-L-lysine) with streptavidin, but these first experiments showed relatively large experimental scatter with respect to the film build-up and to structural data. Nevertheless, it was possible to pattern films by photoablation and to show area-selective binding of fluorescently labeled streptavidin. The combination of electrostatic and specific interactions for film assembly allows for the preparation of molecularly layered multicomposite films with a high degree of complexity (e.g. fine-tuned layer spacings,

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Layer-by-layer protein/polymer hybrid films: T. Cassier et al. incorporation of dye layers, colloid layers\ or layers of several charged proteins\ or DNA) and the consecutive simple attachment of streptavidin to its surface. Complex multilayers with a final layer of streptavidin additionally offer interesting perspectives, because streptavidin will bind any biotinylated material and thus additionally allows for the immobilization of a multitude of functional molecules, even on supports with a complex architecture. However, the experiments described here are, for reasons of simplicity, carried out with a rather primitive film architecture. Therefore, films consist of a precursor film composed of a few layers of the polyelectrolytes poly(styrenesulfonate) PSS and poly(allylamine hydrochloride) PAH, a system which is well investigated, onto which the PLB/streptavidin layers are deposited later on. This type of film architecture also facilitates investigations by X-ray reflectivity, because thicker films yield more Kiessig fringes and values for film thickness and surface roughness are obtained with better accuracy. In this report we concentrate on three topics: (a) optimization of preparation conditions for the build-up of films composed of directly alternating layers of streptavidin and PLB; (b) using the specific interaction between PLB and streptavidin as a tool for the investigation of polyelectrolyte layer interpenetration and (c) controlling the surface roughness of each protein layer and the distance between protein layers by inserting a some spacer layers of flexible polyelectrolytes between the protein layers. In topic (b), we basically test how many non-biotinylated layers are required to cover a PLB layer in such a way that the biotin groups of the ‘‘covered’’ layer are no longer accessible to the streptavidin, in topic (c), we further increase the complexity of the film architecture exploiting the capability of PLB to bind by electrostatic and by specific interaction.

MATERIALS AND METHODS Poly(styrenesulfonate sodium salt) (PSS) and poly (allylamine hydrochloride) (PAH) were obtained from Aldrich. For further purification, PSS was thoroughly dialysed against Milli-Q water and freeze-dried. Poly (L-lysine) (PL) was obtained from Bachem-Biochemica. The synthesis of poly(L-lysine-co-e-biotinyl-L-lysine) was done by biotinylation of poly(L-lysine) using standard methods as follows. In a polymeranalog reaction a solution of the active ester biotin-N-hydroxy-succinimide in chloroform/isopropanol (1 : 1) was added dropwise to a solution of poly(L-lysine ) HBr) in a mixture of triethylamine in methanol. The ratio of triethylamine of e-aminogroups was 4 : 5 and the ratio of active ester to e-aminogroups was 1 : 2. After 30 h of reaction time the turbid suspension was centrifuged at 6000 rpm, the precipitate was washed with methanol and subsequently with chloroform, dried and dissolved in water. The water-insoluble fraction was discarded. The water-soluble fraction was dialyzed against pure water for two days and then lyophilized. The

yield of biotinylated poly(L-lysine) was about 60%, the degree of biotinylation of e-amino groups was about 50% as judged from H-NMR measurements. Streptavidin (SA) was from Boehringer Mannheim. Ultrapure water for all experiments and cleaning steps was obtained by reversed osmosis (Milli-RO 35TS, Millipore GmbH) followed by ion-exchange and filtration steps (Milli-Q, Millipore GmbH). The resistivity was higher than 18 M) z cm and the total organic content less than 10 ppb (according to manufacturer). Glass substrates were from the Gebru¨der Rettberg OHG, Germany. The glass substrates were washed in chloroform in order to remove organic material from the surface. Then they were dipped in a solution of 1% potassium hydroxide in an ethanol/water mixture (v/v"7/3) for 20 min at 60°C and thoroughly rinsed with water. For substrate functionalization a layer of poly(ethyleneimine) (PEI) was adsorbed from a solution of 0.02 monoM PEI in water (monomol"mols of monomer repeat units, in the case of PEI this refers to —(CH —CH —NH)— fragments).   The films were grown by immersion of the substrate in the aqueous solutions of the polymers and streptavidin. The compositions of the solutions for the different films are given in Table 1. Generally, the substrates were washed three times with water after every adsorption step. For the preparation of PSS/PAH precursor films the functionalized substrate was alternately immersed in the PSS/PAH solutions for 20 min beginning with PSS. If not described in another way each precursor film contains four layers PSS and three layers PAH and was terminated with PSS. This corresponds to the following film architecture for the precursor film: substrate/ PEI/(PSS/PAH) /PSS.  The alternating adsorption was carried out by immersion of the substrate in solutions of PLB (30 min) and streptavidin (40 min). For the following experiments the film was dried in a stream of nitrogen and the film thickness was determined by SAXS before and after each streptavidin adsorption step. The experiments for the investigation of polyelectrolyte layer interpenetration were carried out as follows. On a substrate which was coated with a PEI/(PSS/PAH) /PSS precursor film a single layer of  PLB was adsorbed. This substrate was broken into four pieces and those were alternately immersed in solutions of PSS (20 min) and PL (20 min). This way four samples were prepared with an increasing number of spacer layers from 1 PSS layer up to the sequence (PSS/PL) . Then the  different substrates were immersed in a streptavidin solution (30 min) and the thickness of the bound streptavidin layer was determined by X-ray reflectivity. Superlattices of protein/polymer films in which the streptavidin sheets are spacered by the polymer sequences (PLB/PSS/PLB) are prepared in the following way: The precursor film was covered with the sequence [PLB/PSS/PLB] by immersion in aqueous solutions of PLB (30 min), PSS (20 min) and PLB (30 min). For multilayer preparation the substrate was immersed in

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Layer-by-layer protein/polymer hybrid films: T. Cassier et al. Table 1 Composition of solutions for the fabrication of different multilayer films (note that monomol refers to the monomer unit of the respective polymer) Concentrations used for the individual experiments Composition of the solutions used for the individual experiments PSS from 2 mol l\ NaCl PSS from pure water PAH from 2 mol l\ NaCl PLB from pure water PLB from 0.4 mol l\ NaCl PLB from 10 mM l\ octylglucoside Streptavidin from 0.5 mol l\ NaCl Streptavidin from pure water Poly(L-lysine) from 0.06 N HCl

Direct (PLB/streptavidin) alternation

Cover layers

Spacer layers

0.01 monomol l\ — 0.01 monomol l\ — —

—

—

— —

0.01 monomol l\ —

— 0.2 mg ml\ PLB

0.2 mg ml\ PLB —

0.01 monomol l\ — 0.01 monomol l\ 0.2 mg ml\ PLB —

—

0.2 mg ml\ PLB

—

—

— —

10\ mo l\ —

— 10\ mol l\

— 10\ mol l\

—

—

0.17 mg ml\

—

Precursor film

solutions of streptavidin (40 min), PLB (30 min), PSS solution (20 min) and in PLB (20 min) again. In the case of (PLB/PSS/PAH/PSS/PLB) spacer layers the additional PSS and PAH layers were all adsorbed for 20 min. The adsorption of streptavidin and the polymer sequence is repeated in a cyclic fashion and leads to superlattice assemblies. The multilayer buildup was monitored by X-ray reflectivity after drying of the samples. Film thicknesses and surface roughnesses were obtained from fitting experimental curves to a film model consisting of a substrate and a single box for the whole organic layer.

RESULTS AND DISCUSSION Multilayer films by direct alternation of streptavidin and P¸B The driving force for the adsorption of streptavidin layers on PLB and vice versa is the specific recognition reaction of biotin and streptavidin. The repetition of the PLB and streptavidin adsorption in a cyclic fashion should therefore lead to a multilayer assembly (Figure 1). First experiments of PLB/streptavidin film fabrication showed that the growth of such films is possible, but with poor reproducibilities. One object of the present study was to optimize the deposition conditions in order to improve the reliability of the film build-up and to obtain multilayer films of PLB and streptavidin in which the thickness increments per layer are constant over many deposition cycles. A prerequisite for the improvement is a refunctionalization of the surface by biotin groups and streptavidin binding pockets after each adsorption step in such a way that biotin groups on the surface are accessible to the adsorbing streptavidin and vice versa. Note, however, that simple attachment of the streptavidin on the surface is not sufficient to provide for multilayer build-up. The streptavidin must be attached in

such a way that at least one of its four binding pockets remains empty and exposed to the solution interface. If these conditions are not met, that is if the number of available binding sites on the surface increases or decreases with the number of layers deposited, the film growth will either diverge ("overshoot) or terminate ("stagnation). In order to clarify the importance of biotin surface density for streptavidin binding the biotin surface density is estimated. For this purpose a complete surface coverage with PLB and an area requirement of 31.0 nm per streptavidin molecule are assumed. If a biotinylated sidegroup of PLB requires +0.8 nm and the not biotinylated one+0.6 nm, the maximum density of biotin is approximately one biotin/1.4 nm, which allows the adsorption of a dense streptavidin layer. If only one of ten biotin groups are accessible for streptavidin, which is equivalent to an average biotin surface density of one biotin/14 nm still a dense streptavidin layer can be adsorbed. But if the surface density of accessible biotin decreases to one biotin/35 nm (one out of 25 available biotin groups) the adsorption of a dense streptavidin monolayer will no longer be possible. The accessibility of biotin groups at the solution interface of the film is determined by local polymer conformation (density and length of loops) and surface topology on the length scale of the streptavidin (surface roughness). Since functional groups at polymer/solution interfaces are known to be mobile, the surface structure should be dependent on the solvent to some extent. One can imagine that e.g. the rather hydrophobic biotin groups and/or whole polymer loops might adapt conformations unsuitable for proper streptavidin binding in water. In order to change the PLB-conformation we have varied solvent parameters in three ways: Addition of detergent (1;10\ M octylglucoside), of an organic solvent (DMSO/water v : v"1 : 1 or 1 : 10) and of salt (0.4 mol NaCl). It was found that both detergent and organic solvent did not improve proper streptavidin

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Layer-by-layer protein/polymer hybrid films: T. Cassier et al.

Figure 2 Stagnation of [PLB/streptavidin] multilayer growth for the case of adsorption of PLB from an aqueous solution of 1;10\ M octyl-glucoside. The error for the thickness values is within the size of the symbols. The solid line is just a guide to the eye

Figure 1 Oversimplified schematic of the film deposition process. (1) Adsorption of biotinylated poly(L-lysine) (PLB) on a precursor multilayer composed of a few layers of PSS and PAH. (2) Adsorption of streptavidin to biotin groups exposed to the solution interface of the film. Multilayer films are grown by cyclic repetition of steps (1) and (2). Note that this drawing should only help to visualize the multilayer build-up, it is not intended to be an exact representation of polymer conformation and protein orientation within the film

binding. As an example, the stagnation of multilayer growth in the case of PLB adsorption from octylglucoside solution is shown in a plot of the total film thickness versus the layer number (Figure 2).

A large improvement of streptavidin binding was seen after addition of salt to the solution of the PLB, which suggests that the improvement of the presentation of biotin groups at the interface might be due to electrostatic effects. Note that we have kept the composition and ionic strength of the streptavidin solution constant in order not to induce conformational changes in the protein. Owing to the screening effects of the added salt, one would expect the PLB to become more flexible and to form more loops when adsorbed. In this case streptavidin/PLB multilayer assemblies containing, e.g. eight streptavidin layers could be prepared very reproducibly. Figure 3 shows X-ray reflectivity curves during the build-up of a PLB/streptavidin multilayer film. Fitted X-ray data give the growth increment and the surface roughness for each layer. The average thickness of the streptavidin layers is of about 5.3 nm, which is in good agreement with the dimensions of the protein. The projected length of the streptavidin tetramer on its molecular axis are 5.4;5.8;4.8 nm. We found streptavidin layer thicknesses between 4.7 and 6.0 nm, which is in good agreement with the molecular dimensions of the streptavidin and might reflect different orientations of the bound protein on the surface. This is not unlikely if one considers the flexibility of the PLB chains which should allow to bind streptavidin in variable orientations. Figure 4a shows a steady increase of thickness with the number of polymer and protein layers. In Figure 4b the surface roughnesses are plotted versus the layer number. The surface roughness increases with the layer number, but seems to get constant at a limiting value of 2.0—2.5 nm. In spite of the experimental error one can state a change from smoother to rougher surfaces due to the adsorption from streptavidin on PLB and a small smoothing effect for the adsorption of PLB on streptavidin. This reflects the compact and rigid structure of the protein in contrast to the flexibility of the polyelectrolyte in aqueous solutions containing salt. Despite of the encouraging improvement on the reliability of the multilayer formation by direct alternation of

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Layer-by-layer protein/polymer hybrid films: T. Cassier et al.

Figure 3 X-ray reflectivity curves measured during the build-up of a PLB/streptavidin multilayer (adsorption from slat solutions). Note that measurements were done with a small density of data points in order to keep the measuring time short. The solid lines are just connecting the data points

streptavidin and PLB, we cannot offer a detailed structural model which would account for the observed effects. What has been measured is that the thickness of a single PLB layer increases from 0.1 to 0.5 nm in the absence of salt to 1.3 to 2.0 nm in a solution containing 0.4 mol NaCl. This means that the adsorbed amount of PLB increases about 5-fold. However, we can only speculate if the improved streptavidin binding is due to the increased amount of biotin (unlikely) or to changes in conformation and accessibility (likely). A third possibility would be that the salt in the PLB solution favors a PLB conformation that does not allow the blocking of all four binding sites of the streptavidin by binding to the underlying layer (unlikely). However, a layered structure of the streptavidin/PLB films can be derived if one considers the linear growth of the film and regards the change between smooth and rough surface after adsorption of the flexible PLB chains and the compact streptavidin molecules. ¹he specific P¸B/streptavidin interaction used as a tool for the investigation of polyelectrolyte layer interpenetration The property of PLB to build up multilyer by two different interactions was ued to probe the interpenetration depth of adjacent layers. Four (PSS/PAH)-precursor films terminated with one monolayer of PLB were covered with an increasing number of additional layers from 1 PSS layer up to the sequence (PSS/PL) ("cover 

Figure 4 (a) Increase of film thickness with the number of layers of streptavidin and PLB in the case of adsorption from salt solutions. The error for the thickness is within the size of the symbols. (b) Increase of the surface roughness during the multilayer growth of PLB and streptavidin by adsorption from salt solutions. Note that the roughness is constant (+2 nm) after a few layers. For reasons of clarity the error bars are not shown in the plot. The solid lines are just a guide to the eye.

layers). It was tested whether streptavidin can bind to the surface of these films although the PLB-layer was covered by additional polymer layers. This is only possible if layer interpenetration, that is loops of a single chain stretching across several layers deposited above, allows streptavidin to bind to the PLB in an underlying layer. The film thicknesses of the streptavidin and the polyelectrolyte cover layers are plotted versus the number of covering layers in Figure 5. The streptavidin layer thickness decreases from 5.3 nm (no cover layer) to 3.99 nm with one PSS cover layer and further down to 1.62 nm with four cover layers of PSS and PL. Thus, it is clearly visible that a small amount of streptavidin (about 30%) still was adsorbed when four cover layers with a thickness of 3.48 nm were deposited above the PLB layer. This is in agreement with the recently reported model for a polyelectrolyte multilayer film composed of highly charged flexible polyelectrolytes assuming a 1 : 1 stoichoimetry. This strong overelap with neighboring layers is also in agreement with results obtained earlier from repeated adsorption of negatively charged PSS on the surface of a (PSS/PAH) multilayer which had a layer of PSS as last layer on the surface. In this previous work the initial thickness of the terminating PSS-layer

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Figure 5 Test of layer interpenetration. In the plot is shown the dependence of streptavidin and spacer layer thicknesses in dependence on the number of polyelectrolyte spacer layers. The solid lines are just a guide to the eye

was 4.5 nm, but three additional PSS layers could subsequently be adsorbed directly onto this last PSSlayer yielding a final thickness of more than 10.0 nm for this PSS layer. Note that this does not necessarily imply that PSS adsorbs to a surface already covered with PSS. This is in fact not the case since the adsorption of each polyion layer is self-regulating in the sense that once the surface potential has been reversed, newly arriving chains of the same charge are blocked from adsorbing on the surface. Only when the multilayer is washed in pure water and dried, one has to assume that the surface reorganizes due to the change in hydration conditions. The repetition of PSS adsorption requires the presence of PAH segments for the PSS to bind to, so this result suggests a layer interpenetration in a similar way as shown here for the PLB/cover layer/streptavidin system.

From the plots of the film thickness (Figure 6a) and the surface roughness (Figure 6b) versus the layer number one can state an increase of the thickness increments of the [PLB/PSS/PLB] sequences and of the surface roughness. After adsorption of three repeat unit the Kiessig fringes in the X-ray curves disappeared caused by the increased surface roughness, which makes the determination of film thickness impossible. Here obviously we have enough well-exposed binding sites for streptavidin after the deposition of the [PLB/PSS/PLB] spacer sequence, but the surface roughness overshoots when the PLB and the streptavidin are adsorbed from pure water in which the PLB is considerably less flexible that in slat solution. In order to prevent increasing surface roughness we have used additional spacer layers of PSS and PAH, both deposited from salt solutions, which can be expected to have a smoothing effect. In this way a complex multilayer system of alternating [PLB/PSS/PAH/PSS/PLB] and streptavidin layers sequences was built up to seven protein/polymer repeat units as shown in Figure 7a. During the multilayer deposition the surface roughness increased up to the fourth streptavidin layer and then fluctuated

More complex film architectures: tuning of streptavidin layer separation by polyelectrolyte spacer layers The spacing between the streptavidin sheets can be tuned by insertion of varying numbers of polyelectrolyte layers ("spacer layers). These complex multilayer systems can be fabricated, because PLB can bind to neighboring layers both by electrostatic and by specific interaction. The insertion of spacer layers also allows to control the surface roughness of the film. In order to show the importance of the surface roughness we have not used the optimized deposition conditions that were used for the direct alternation of PLB and streptavidin, but the less reliable adsorption in the absence of salt. In this case properties of the surface layer such as its interfacial roughness have a more pronounced influence on the film build-up. We show two examples of multilayer films, in which spacer sequences of [PLB/PSS/PLB] and [PLB/PSS/PAH/PSS/PLB] are inserted between the streptavidin layers. In the first example we investigated the insertion of one PSS and two PLB layers to get a multilayer system of [PLB/PSS/PLB] sequences alternated with streptavidin.

Figure 6 (a) Increase of film thickness with the number of layers of streptavidin and PLB in the case of alternation of streptavidin and [PLB/PSS/PLB] spacer sequences. The error for the thickness is within the size of the symbols. The solid line is just a guide to the eye. (b) Increase of the surface roughnesses during the multilayer growth of PLB and streptavidin in the case of alternation of streptavidin and [PLB/PSS/PLB] sequences. The solid line is a linear fit to the roughness data and shows that the roughness is constantly increasing as more layers are deposited

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Layer-by-layer protein/polymer hybrid films: T. Cassier et al. solutions containing salt, whereas addition of detergents or organic solvents had no beneficial effects. The average thickness of the streptavidin layers is about 5.3 nm, which is in good agreement with the dimensions of the protein. The interpenetration of adjacent layers was demonstrated by the fact that streptavidin even binds to films whose PLB layers were covered by several layers of non-biotinylated polymer. Furthermore, we have shown that the distance between the protein sheets can be tuned by insertion of a variable number of polyelectrolyte spacer layers. An example of such a multilayer system, in which the sequence [PLB/PSS/PAH/PSS/PLB] is inserted between the streptavidin layers, leads to an average distance of 6.5 nm between the protein sheets. The experiments described here suggest that structural film parameters such as the surface roughness play a pronounced role in the multilayer build-up. If such parameters are properly controlled, a reliable multilayer build-up is even possible by employing interactions that have a high steric demand. REFERENCES

Figure 7 (a) Increase of film thickness with the number of layers of streptavidin in the case of alternation of streptavidin and [PLB/PSS/PAH/PSS/PLB] spacer sequences. The error for the thickness is within the size of the symbols. (b) Increase of the surface roughness during the multilayer growth of streptavidin and [PLB/PSS/PAH/PSS/PLB] as a spacer sequence. Note that the roughness gets constant (+1.5 nm) after a few layers. The solid lines are just a guide to the eye

around a constant value of 1.4 nm, but did not overshoot (Figure 7b). The fitted X-ray data lead to an average spacing between streptavidin layers of 6.5 nm. Astonishingly, the individual layer thicknesses for the streptavidin layers are only 3.2$0.7 nm, which is considerably smaller than the dimensions of the protein. Most likey, the surface of the polyelectrolyte spacer layers is so soft that the protein does not just adsorb on the surface, but can sink in to some extent. Such an embedding is not unlikely given the surface roughness of 1.4 nm and has also been observed in the deposition of virus particles on top of a polyelectrolyte multilayer. Since good film growth was already achieved in this way, it was not necessary to apply a third method of additional multilayer smoothing which consists of consecutive immersion of polyelectrolyte films in salt solution and in water.

CONCLUSIONS Layer-by-layer adsorption can also be carried out employing specific interactions between molecules. The reliability of multilayer construction with streptavidin/ PLB has been improved by depositing the PLB from

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